Cellobiohydrolases Produce Different Oligosaccharides from Chitosan

Abstract

Chito-oligosaccharides (COSs) are bioactive molecules with interesting characteristics; however, their exploitation is still restricted due to limited amounts accessible with current production strategies. Here we present a strategy for the production of COSs based on hydrolysis of chitosan by using readily available glycosidases. Cellobiohydrolases (EC 3.2.1.91) were compared with chitosanases (EC 3.2.1.132) regarding their ability for COS production, and the resulting fractions were analyzed by MS and NMR. The oligosaccharides had a degree of polymerization between three and six units, and the degree of acetylation (DA) varied depending on the applied enzyme. Different cellobiohydrolases produced COSs with varying DA, and based on comprehensive NMR analysis the preferred cleavage sites of the respective enzymes that show chitosanase and chitinase activity were elucidated. The study reveals the high potential of readily available cellulolytic enzymes besides chitosanases for the production of COSs with distinct structure facilitating access to this bioactive compound class.

Crude Enzyme Concentrate of Filamentous Fungus Hydrolyzed Chitosan to Obtain Oligomers of Different Sizes

“…Two cellobiohydrolases were identified in the present work: Probable 1,4-β-D-glucan cellobiohydrolase A (Q5B2Q4) and Probable 1,4-β-D-glucan cellobiohydrolase B (Q8NK02). These enzymes release cellobiose units from cellulose's reducing and non-reducing ends [56]. Cellobiohirolases can also hydrolyze chitosan, producing low molecular weight oligomers.…”

Biochemical Characterization of a GH46 Chitosanase Provides Insights into the Novel Digestion Specificity

“…1 However, multiple applications and further investigations of chitosan in various areas are limited due to its poor water solubility. 2 A more ecological, simple, valuable, and efficient utilization is the conversion of chitosan biomass to chito-oligosaccharides (COSs) and their monomer (GlcN), 3,4 which exhibit unique functionalities and bioactive properties. Furthermore, COSs and GlcN have been implemented in a variety of applications like food, pharmaceutics, cosmetics, and agriculture.…”

“…Chitosan, a biopolymer with a random distribution of β-(1,4)-linked N -acetyl- d -glucosamine (GlcNAc, A) and d -glucosamine (GlcN, D) units, is considered to be among the versatile and promising functional biopolymers . However, multiple applications and further investigations of chitosan in various areas are limited due to its poor water solubility . A more ecological, simple, valuable, and efficient utilization is the conversion of chitosan biomass to chito-oligosaccharides (COSs) and their monomer (GlcN), , which exhibit unique functionalities and bioactive properties.…”

Quantitative Mass-Spectrometric Sequencing of Chitosan Oligomers Revealing Cleavage Sites of Chitosan Hydrolases

“…Powerful tools for analyzing and modifying chitosans are chitinase and chitosanase enzymes cutting chitosans at specific sequence motifs, but for these enzymes to be useful, both for the development of fingerprinting analyses and for the production of defined partially acetylated chitosan oligosaccharides (paCOS), precise knowledge of their subsite specificities is required. ,, Unfortunately, we know these for only a few chitosan hydrolases, mostly because so few analytical tools exist that allow detailed analysis of these enzymes’ polymeric substrates and oligomeric products, especially when only small amounts of these compounds are available . For example, the conventional analytical method, size-exclusion chromatography followed by nuclear magnetic resonance (SEC-NMR), requires many milligrams of sample, only separates oligomers according to their DP, and only gives average values of the DA and of the occurrence of GlcN or GlcNAc subunits at or near the reducing and nonreducing ends of the oligomers. , Also, SEC-NMR is time-consuming, but rapid analyses are needed, e.g., for screening steps in evolutionary optimizations of chitosan modifying enzymes.…”

“…12 For example, the conventional analytical method, size-exclusion chromatography followed by nuclear magnetic resonance (SEC-NMR), requires many milligrams of sample, only separates oligomers according to their DP, and only gives average values of the DA and of the occurrence of GlcN or GlcNAc subunits at or near the reducing and nonreducing ends of the oligomers. 13,14 Also, SEC-NMR is time-consuming, but rapid analyses are needed, e.g., for screening steps in evolutionary optimizations of chitosan modifying enzymes.…”